Telomeres are the physical ends of chromosomes that protect genome stability by preventing chromosomes from being damaged. Normal somatic cells have limited lifespan due to the loss of telomere DNA during cell proliferation. Understanding the mechanisms that protect telomeres from rapid shortening is vital for preventing premature aging. Studies have discovered that excessive telomere shortening can be attributed to defective C-strand fill-in, telomere replication stress, and aberrant telomere chromatin structure. However, the mechanisms underlying these three pathways are poorly understood. Recently, the Ctc1/Stn1/Ten1 (CST) single-stranded DNA binding complex has been identified as a new player in protecting telomeres. Mutations in Ctc1 cause a complex disease known as Coats Plus, and cells derived from Coats Plus patients display short telomeres. We have found that deficiency in components of the CST complex causes catastrophic telomere loss, inducing early cellular senescence and DNA damage response in human somatic cells. Therefore, it is important to understand the functions of CST in preventing telomere loss. Our data suggest that the CST complex is a multifunctional protein that promotes efficient replication of telomeric DNA as well as mediates C-strand fill-in at chromosome ends to counteract excessive telomere shortening. In addition, our data also suggest that the CST complex may be involved in regulating telomeric chromatin structure, which is critical for maintaining telomere length. The objective of this proposal is to determine the roles of the CST complex in preventing excessive telomere loss, so that effective strategies for treating premature aging associated with telomere loss can be developed. Experiments are designed to determine how post-translational modification of CST facilitates telomeric DNA replication and precisely regulates C-strand fill- in (Aims 1 and 2). In addition, we will determine the effect of replication fork stalling on telomere chromatin organization (Aim 3). This will be accomplished by integration of mass spectrometry, fluorescent DNA fiber analysis, and molecular/biochemical methods. Completion of the proposed research is expected to substantially add to our understanding of telomere maintenance. It may lead to new directions in studying telomere maintenance and facilitating the development of anti-aging therapy.